Nucleic acid molecule encoding abscisic acid responsive element-binding factor 2

ABSTRACT

A nucleic acid molecule encoding the Abscisic acid responsive element binding factor 2 (ABF2) was isolated and its nucleotide sequence determined. ABF2 belongs to the ABF family of factors which bind abscisic acid responsive elements in plants. Expression of ABFs is inducible by abscisic acid and various stress treatments. ABFs have the potential to activate a large number of abscisic acid/stress responsive genes and thus a nucleic acid molecule encoding ABF2 can be used to generate transgenic plants that are tolerant to multiple environmental stresses.

This application is a division of Ser. No. 09/416,050 filed Oct. 12,1999.

BACKGROUND OF THE INVENTION

This invention relates to a family of novel transcription factors thatbind to various abscisic acid responsive elements(ABREs), moreparticularily, factors named as ABFs(ABRE-Binding Factors) isolated byyeast one-hybrid screening of an Arabidopsis cDNA expression libraryusing a prototypical ABRE (SEQ ID NO: 9; GGACACGTGGCG).

Abscisic acid (ABA) is one of the major plant hormones that plays animportant role during plant growth and development (Leung and Giraudat,1998). The hormone controls several physiological processes during seeddevelopment and germination. During vegetative growth, ABA is known tomediate responses to various adverse environmental conditions such asdrought, high salt and cold/freezing (Shinozaki and Yamaguchi-Shinozaki,1996).

One of the ABA-mediated responses to various environmental stresses isthe induced expression of a large number of genes, whose gene productsare involved in the plant's adaptation to the stresses (Ingram andBartels, 1996). ABA responsive elements (ABREs), i.e., cis-regulatoryelements that mediate the ABA-modulated gene expression, have beenidentified from the promoter analysis of ABA-regulated genes (reviewedin Busk and Pages, 1998). One class of the ABREs includes elements thatshare a PyACGTGGC (Py indicates C or T) consensus sequence, which can beconsidered a subset of a larger group of cis-elements known as “G-box”(Menkens et al., 1995). Another class of ABREs, known as “couplingelements (CE)” or “motif 111”, shares a CGCGTG consensus sequence. Bothclasses of ABREs, here, referred to as G/ABRE (G-box-like ABRE) andC/ABRE (CE-like ABRE), respectively, are almost ubiquitous in thepromoter regions of ABA responsive genes of both monocotyledonous anddicotyledonous plants.

A number of basic leucine zipper (bZIP) class DNA-binding proteins areknown to interact with the ABREs (Busk and Pages, 1998). EMBP1 and TAF1have been isolated based on their in vitro binding activity to G/ABREs.GBF3, originally identified as one of the G-box binding factors (GBFs)involved in the light regulation of a ribulose bisphosphate carboxylasegene (Schindler et al., 1992), has been cloned using the ABA-responsive,G-box element of a Arabidopsis Adh1 gene. Recently, a family ofembryo-specific factors has been reported that can recognize both G/ andC/ABREs (Kim and Thomas, 1998; Kim et al., 1997). Other factors bindingto G-box have also been described (Foster et al., 1994).

Although ABRE-binding factors have been known for some time, severalobservations suggest that hitherto unidentified factors are involved inABA-regulated gene expression during stress response, especially invegetative tissues. ABA-induction of rice rab16A and Arabidopsis rd29Bgenes requires de novo protein synthesis (Nakagawa et al., 1996;Yamaguchi-Shinozaski and Shinozaki, 1994), suggesting the involvement ofABA-inducible factors. In vivo binding of ABA-inducible factors has beendemonstrated in maize rab17 gene (Busk et al., 1997). In the case ofrab16B gene, currently unknown, C/ABRE-binding factor(s) has beensuggested to mediate ABA response through the motif III (Ono et al.,1996). Furthermore, it has been well established by genetic studies thatdifferent ABA signaling pathways operate in seeds and in vegetativetissues, respectively (Leung and Giraudat, 1998), and tissue-specificABRE-binding activities have been demonstrated (Pla et al., 1993). Noneof the source materials used in the previous protein-DNA interactionclonings, however, were ABA- or stress-treated young plant tissues, andthus, inducible factors that may be critical for the ABA-mediated stressresponse during vegetative growth phase may have been missed so far.

Numerous stress responsive genes involved in plant's adaptation tovarious environmental stresses are regulated by ABA through G/ABREs orC/ABREs (Ingram and Bartels, 1996). Therefore, overexpression ofABRE-binding transcription factors will result in the activation ofthese stress-inducible genes and thus enhanced stress tolerance. Hence,once isolated, the ABRE-binding factors will be suitable for thegeneration of transgenic plants that are tolerant to multipleenvironmental stresses. Feasibility of manipulating transcriptionfactors for the improved stress tolerance has been demonstrated byothers recently (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999).

SUMMARY OF THE INVENTION

This invention relates to a family of novel transcription factors thatbind to various ABREs. The factors, named as ABFs (ABRE-BindingFactors), were isolated by yeast one-hybrid screening of an ArabidopsiscDNA expression library using a prototypical ABRE (SEQ ID NO: 9;GGACACGTGGCG). ABFs are bZIP class transcription factors that can bindto both G/ABREs and C/ABREs. Expression of ABFs is inducible by ABA andvarious stress treatments, and they can transactivate an ABRE-containingreporter gene in yeast. Thus, ABFs have potential to activate a largenumber of ABA/stress responsive genes and thus can be used to generatetransgenic plants that are tolerant to multiple environmental stresses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Specificity of ABF binding. Binding specificity of ABFs. Areporter yeast containing a HIS3 reporter construct with (+) or without(−) the ABRE (a trimer of Emla; SEQ ID NO: 10) was transformed with DNAfrom representative clones, and transformants were grown on eithergalactose (GAL) or glucose (GLU) medium lacking histidine.

FIGS. 2A-2D. Nucleic and deduced amino acid sequences of ABFs. Nucleicacid sequences of ABFs are presented together with deduced amino acidsequences. The bZIP and the glutamine-rich regions are underlined. FIG.2A: ABF1 (SEQ ID NO: 1 and 2), FIG. 2B: ABF2 (SEQ ID NO: 3 and 4), FIG.2C: ABF3 (SEQ ID NO: 5 and 6) and FIG. 2D: ABF4 (SEQ ID NO: 7 and 8)correspond to clones 1, 2, 11, and 19, respectively.

FIG. 3. Alignment of the deduced amino acid sequences of ABFs. Thededuced amino acid sequences of ABFs (SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8) are aligned together. The basic region and theleucine repeats are shown by a thick line and arrowheads, respectively.The small arrow indicates the arginine and the lysine residues withinthe basic regions that are discussed in the text. Regions highlyconserved are highlighted. #, CaMK II sites. +, CK II sites.

FIGS. 4A-4B. Electrophoretic mobility shift assay. FIG. 4A, ABF1 bindingto a G/ABRE. An oligonucleotide (ABRE) containing the Em1a element (SEQID NO: 11) was employed as a probe in a mobility shift assay. FIG. 4B,ABF1 binding to a C/ABRE. An oligonucleotide containing the hex-3sequence (SEQ ID NO: 13) was employed as a probe. In each assay, 1 μg ofrecombinant ABF1 was used. Lanes 1, probe only (−); 2, probe ABF1 (+); 3and 4, 100-fold and 200-fold molar excess of specific competitors (ABRE,SEQ ID No: 13 and hex-3, SEQ ID NO: 13), respectively; 5 and 6, 100-foldand 200-fold molar excess of a mutated oligonucleotide (mABRE, SEQ IDNo: 12 and mhex-3, SEQ ID NO: 14) as competitors, respectively.Sequences of oligonucleotides are shown at the bottom of each figure andshifted bands are indicated by arrowheads.

FIGS. 5A-5B. Binding site selection assay. FIG. 5A, binding siteselection assay. Top; Probes (P0 to P5) after each round of selectionwere amplified and used in EMSA. 1.5 μg of ABF1 was used. Only the toppart of the gel containing the shifted bands is shown. Bottom; EMSA ofP5 probe DNA. P5 probe DNA was employed in EMSA and titrated withincreasing amount (μg) of ABF1. Arrowheads denote shifted bands. Theband shown by * is probably an artifact resulting from secondarystructure formation of palindromic sequences in the selected sequencesFIG. 5B, selected sequences. The selected sequences (SEQ ID NOS: 33-79)are aligned and grouped according to their consensus sequences shown inparentheses. The nucleotides highly conserved within each group are inbold, and those 100% conserved are underlined. G/ABRE elements flankingthe C/ABRE core of group II sequences are in italics and underlined. Thenumber of selected sequences in each group is indicated in theparentheses on the right.

FIGS. 6A-6B. Analysis of ABF expression. ABA- and stress-inducibility ofABF expression were examined by RNA gel blot analysis or RT-PCR. A,inducibility of ABF expression. 25 μg of total RNAs isolated fromuntreated plants or plants treated with ABA, high salt, cold or drought,were transferred to a membrane and probed with specific probes. B, timecourse of ABA-induction. RT-PCR reactions were performed using 0.5 μgtotal RNAs from plants treated with 100 μM ABA for 0 min, 30 min, 1 hr,2 hr, 4 hr, 8 hr 12 hr, 16 hr, and 24 hr. actin, a control reactionperformed with an actin gene of Arabidopsis thaliana.

FIG. 7. Transactivation assay of ABFs. Transactivational function ofABFs was tested by using a yeast system. ABFs were expressed in yeastthat harbored an ABRE-containing lacZ reporter gene. The β-galactosidaseactivity was then assayed and indicated as Miller units. For eachconstruct, 5 different transformants were assayed in duplicates. YX243,control vector without any inserts.

FIGS. 8A-8B. Phylogenetic analysis of ABRE-binding bZIP factors. FIG.8A, bZIP regions of the ABRE factors mentioned in the text (SEQ ID NO:80, SEQ ID NO: 81, SEQ ID NO. 82, SEQ ID NO: 83). mlip15 is a maize bZIPfactor induced by low-temperature (SEQ ID NO: 28; Kusano et al., 1995).Conserved amino acids are highlighted, and the leucine residues in the“zipper” regions are underlined (SEQ ID NOs: 2, 4, 6, 8 and DPBF1, SEQID NO: 25; DPBF2, SEQ ID NO: 26; DPBF3, SEQ ID NO: 27; mlip15, SEQ IDNO: 28; EMBP1, SEQ ID NO: 29; OSBZ8, SEQ ID NO: 30; GBF3, SEQ ID NO: 31;TAF1, SEQ ID NO: 32-25-32). FIG. 8B, unrooted phylogenetic tree diagram.The bZIP regions shown in FIG. 8A. The bZIP regions shown in A werealigned and a tree diagram was constructed using CLUSTAL W algorithm.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods

Plant materials—Arabidopsis thaliana (ecotype Columbia) was grown at 22°C. on pots of soil (a 1:1 mixture of vermiculite and peat moss)irrigated with mineral nutrient solution (0.1% Hyponex) in 8 hr light/16hr dark cycles. For RNA isolation, 4-5 weeks old plants were subject tovarious treatments, flash-frozen in liquid nitrogen and kept at −70° C.until needed. For ABA treatment, roots of plants were submerged, afterthe removal of soil, in a 100 μM ABA (Sigma, No A 1012) solution for 4hr with gentle shaking. ABA solution was also sprayed intermittentlyduring the incubation period. Salt treatment was performed in the sameway, except that 250 mM NaCl solution was employed. For droughttreatment, plants were withheld from water for two weeks before harvest,and left on the bench, after removing soil, for 1 hr just beforecollection. For cold treatment, plants were placed at 4° C. for 24 hrunder dim light before harvest.

Yeast techniques, DNA manipulation and RNA gel blot analysis—Standardmethods (Ausubel et al., 1994; Guthrie and Fink, 1991; Sambrook et al.,1989) were used in manipulating DNA and yeast. DNA sequencing wasperformed on ABI 310 Genetic Analyzer, according to the manufacturer'sinstruction. DNA sequence analysis was done with DNA Strider® andGenerunr®, and BLAST algorithm (Altschul et al., 1990) was used fordatabase search. Multiple sequence alignment and phylogenetic treeconstruction were performed with CLUSTAL W program (Thompson et al.,1994) available on the web (http://www2.ebi.ac.uk/clustalw).

RNA was isolated according to Chomczynski and Mackey (1995) and furtherpurified by LiCl precipitation followed by ethanol precipitation. ForRNA gel blot analysis, 25 μg of total RNA was fractionated on 1.1%formaldehyde agarose gel, transferred to nylon membrane (Hybond-N+,Amersham) by “downward capillary transfer” method (25), and fixed usingStratagene's UV Crosslinker (Model 2400). Loading of equal amount ofRNAs was confirmed by ethidium bromide staining. Hybridization was at42° C. in 5X SSC (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate), 5XDenhardt's solution (1X Denhadt's solution is 0.02% Ficoll, 0.02% PVP,0.02% BSA), 1% SDS, 100 μg/ml salmon sperm DNA, and 50% foramide for24-30 hrs. Probes were prepared from the variable regions of ABFs. Afterhybridization, filters were washed twice in 2X SSC, 0.1% SDS at roomtemperature and three times in 0.2X SSC, 0.1% SDS for 10 min each at 65°C. Exposure time was 7-8 days. RT-PCR was performed employing the AccessRT-PCR System (Promega) using 0.5 g of total RNA according to themanufacturer's instruction. Amplification after the first strand cDNAsynthesis was 45, 35, 40 and 45 cycles for ABF1, 2, 3 and 4,respectively. ABF primers (sequences are available upon request) werefrom variable regions between the bZIP and the conserved regions. Theactin primers used in the control reaction was from the Arabidopsisactin-1 gene (GenBank Accession No., M20016). Free of contaminating DNAin RNA samples was confirmed by using primer sets (ABF3 and actin) thatflank introns.

cDNA library construction and yeast one-hybrid screening-Poly A(+) RNAwas isolated from total RNAs prepared from ABA- or salt-treatedArabidopsis seedlings, using Qiagen's Oligotex resin. cDNA wassynthesized from an equal mixture (6 μg total) of poly A(+) RNAsprepared from the two sources of total RNAs employing a Stratagene'scDNA synthesis kit. cDNA was fractionated on a Sepharose CL-2B column,peak fractions containing cDNAs larger than 500 bp were pooled, andpooled cDNAs were ligated with pYESTrp2 (Invitrogen) predigested withEco RI-Xho I. The ligation mixture was electroporated into E. coli DH10Bcells. Titer of this original library was 5.4×10⁷ cfu. Portion of thelibrary (2×10⁷) was plated on 15 cm plates at a density of 150,000cfu/plate. Cells were suspended in LB after overnight growth at 37° C.on plates and pooled together. Finally, plasmid DNA was prepared fromthe collected cells.

pYC7-I and pSK1 (Kim and Thomas, 1998) were used as HIS3 and lacZreporter plasmids, respectively. The G/ABRE reporter construct wasprepared by inserting a trimer of Em1a element (SEQ ID NO: 9; Guiltinanet al., 1990) into the Sma I site of pYC7-I and the Xba I site of pSK1.In order to prepare reporter yeast, YPH 500 was transformed with the StuI-digested pYC7-I reporter construct. Resulting Ura+ colonies weretransformed with the pSK1 construct and maintained on a SC-LEU-URAmedium. Screening of the library was performed as described (Kim andThomas, 1998) except that transformed reporter yeast was grown onGal/Raf/CM-HIS-LEU-TRP plates instead of Glu/CM-HIS-LEU-TRP plates.Putative positive clones from the screen were streaked on freshGal/Raf/CM-HIS-LEU-TRP plates to purify colonies. After β-galactosidaseassay, well-isolated single colonies were patched on Glu/CM-LEU-TRP-URAplates to be kept as master plates. Galactose-dependency of theHis⁺/lacZ⁺ phenotype of the purified isolates was examined subsequentlyby comparing their growth pattern and β-galactosidase activity onGal/Raf/CM-HIS-LEU-TRP and Glu/CM-HIS-LEU-TRP dropout plates.

Analysis of positive clones—Yeast DNA was prepared from 1.5 ml ofovernight cultures of the positive clones. PCR was performed withprimers derived from the pYESTrp2 vector sequences flanking the inserts(pYESTrp forward and reverse primers). PCR products were digested withEco RI, Hae III, or Alu I in order to group the cDNAs. For libraryplasmid rescue, yeast DNAs from representative clones were introducedinto DH10B E. coli cells by electroporation. Plasmid DNAs used in DNAsequencing and confirmation experiments were isolated from these E. colitransformants by the alkaline lysis method. For the confirmationexperiment shown in FIG. 1, plasmid DNAs thus isolated werere-introduced into the yeast containing pSK1 or ABRE-pSK1, transformantswere kept on Glu/CM-LEU-TRP plates, and their growth was tested afterspotting 5 μl of overnight cultures (1/50 dilutions) onGal/Raff/CM-HIS-LEU-TRP or Glu/CM-HIS-LEU-TRP plates containing 2.5 mM3-aminotriazole.

Isolation of full-length ABF3 and 4—A PCR approach was used to isolatethe missing 5′ portions of clone 11 and clone 19. Database searchrevealed that clone 11 was part of the BAC clone F28A23 of theArabidopsis chromosome IV. On the other hand, the 5′ portion of theclone 19 sequence was identical to the 3′ region of an EST clone,176F17T7. Based on the sequence information, 5′ PCR primers(5′-GAAGCTTGATCCTCCTAGTTGTAC-3′ for clone 11 and5′-ATTTGAACAAGGGTTTTAGGGC-3′ for; SEQ ID NO: 16; for clone 19) weresynthesized. 3′ primers (5′-TTACAATCACCCACAGAACCTGCC-3′; SEQ ID NO: 17and 5′-GATTTCGTTGCCACTCTTAAG-3′; SEQ ID NO: 18, which are complementaryto the 3′-most sequences of clones 11 and 19, respectively) wereprepared using our sequence information. PCR was performed with Pwopolymerase (Boeringer Mannheim), using the primer sets and 1 μg of ourlibrary plasmid DNA. After 30 cycles of reaction, the DNA fragmentscorresponding to the expected size of the full-length clones weregel-purified and cloned into the PCR-Script vector (Stratagene). Severalclones from each PCR product were then sequenced in their entirety. Thefidelity of the full-length sequences was confirmed by comparing theirsequences with each other and with those of the original partial clonesand the genomic clones deposited later by the Arabidopsis GenomeInitiative Project.

Plasmid constructs—In order to prepare a GST-ABF fusion constructs,entire coding regions and the 3′ untranlated regions of ABF1 and ABF3were amplified by PCR using Pfu polymerase (Stratagene). After Xho Idigestion followed by gel-purification, the fragments were cloned intothe Sma I-Sal I sites of pGEX-5X-2 (Pharmacia Biotech). The constructsused in transactivation assay were also prepared in a similar way. Thecoding regions were amplified by PCR. Resulting fragments were digestedwith Xho I, gel-purified and cloned into pYX243. pYX243 was prepared byNco I digestion, Klenow fill-in reaction, Sal I digestion andgel-purification. Intactness of the junction sequences was confirmed byDNA sequencing.

Preparation of recombinant ABFs and mobility shift assay—RecombinantABF1 and ABF3 were prepared employing a GST Purification Module fromPharmacia Biotech, according to the supplier's instruction. E. coli BL21cells were transformed with the GST-ABF constructs by electroporation.In order to prepare bacterial extract, a single colony of transformedbacteria was inoculated in 2YT/Amp medium and grown overnight. Theculture was diluted (1:100) into 250 ml of fresh media. IPTG was addedto the culture to a final concentration of 0.1 mM when A₆₀₀ reached 0.7.Cells were harvested by centrifugation after further growth (1.5 hr).The bacterial pellet was resuspended in 12.5 ml of PBS (0.14 M NaCl, 2.7mM KCl, 10.1 mM Na₂HPO₄, 1.8 mM KH₂PO₄, PH7.3) and sonicated on aBranson Sonifier 250 (4×40 s burst at setting 5 at 80% duty cycle). Thelysate was cleared of cell debris by centrifugation, and the supernatantwas loaded onto a column packed with 0.125 ml (bed volume) ofglutathione Sepharose 4B resin. Wash and elution was performed assuggested by the supplier. Protein concentration was determined usingthe BIO-RAD protein assay kit. Production of GST-ABF1 fusion protein wasconfirmed by Western blotting using GST antibody.

Mobility shift assay was performed as described (Kim et al., 1997). Toprepare probes, oligonucleotide sets shown in FIG. 4 were annealed byboiling 100 pmoles each of complementary oligonucleotides for 5 min andslowly cooling to room temperature. Portions of the annealedoligonucletides (4 pmoles of each set) were labeled by Klenow fill-inreaction in the presence of ³²P-dATP. Binding reactions were on ice for30 min, and electrophoresis was performed at 4° C.

Binding site selection assay—A pool of 58 bases oligonucleotide, R58,containing 18 bases of random sequence was synthesized:CAGTTGAGCGGATCCTGTCG(N)₁₈GAGGCGAATTCAGTGCAACT (SEQ ID NO: 19). Therandom sequence is flanked by Bam HI and Eco RI sites for theconvenience of cloning after selection. R58 was made double strand byannealing a primer, RANR, (SEQ ID NO: 20) AGTTGCACTGAATTCGCCTC, and thenby extending it using Klenow fragment. For the first round of selection,5 pmoles of the double strand R58 (P0 probe) was mixed with 5 μg of therecombinant ABF1 in a 100 μl of binding buffer (10% glycerol, 25 mMHEPES, pH 7.6, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT) containing 4 μg ofpoly [d(I-C)] and incubated on ice for 30 min. The mixture was loadedonto 0.1 ml of glutathione Sepharose 4B resin packed on a disposablecolumn, washed with 10 volumes of the binding buffer, and eluted with0.3 ml of 10 mM glutathione. Bound DNA was purified by phenol/chloroformextraction followed by ethanol precipitation. Amplification of theselected DNA was performed by PCR, using 20 pmoles each of RANF SEQ IDNO: 21) (CAGTTGAGCGGATCCTGTCG) and RANR primers in a buffer (10 mM Tris,pH 9.0, 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl₂) containing 150 μMdNTP-dATP, 4 μM dATP, 10 μCi of ³²P-dATP. Reaction was carried out 20cycles (10 sec, 94° C./10 sec, 50° C./1 min, 72° C.). Amplified DNA waspurified on a polyacrylamide gel, the band was excised afterautoradiography, and DNA was eluted by standard method to be used as aprobe DNA for the next round of selection. The selection cycle wasrepeated two more times. For the fourth and the fifth rounds ofselection, bound DNAs were isolated after EMSA, by eluting DNA from thedried gel fragment containing the shifted bands. The amplified DNA (P5probe) from the last selection was cloned into pBluescript (Stratagene)after Eco RI and Bam HI digestion, and plasmid DNAs from 50 randomcolonies were sequenced.

Transactivation assay—Reporter yeast containing the lacZ reporter gene(pYC7-1) with or without the ABRE was transformed with variouspYX243/ABF constructs, and transformants were kept on Glu/CM-LEU-URAplates. For the assay, 5 colonies from each transformant group weregrown in a Glu/CM-LEU-URA medium overnight to A₆₀₀ of approximately 1.The cultures were diluted 4-6 times with fresh media, grown further for3 hr, and pelleted by brief centrifugation. The cells were washed twicewith Gal/Raf/CM-LEU-URA medium, resuspended in 4 ml of the same medium,and grown for 4 hr to induce the expression of ABFs. A₆₀₀ was measuredat the end of the growth period, and 0.5 ml aliquots of the culture, induplicates, were pelleted. The pellets were resuspended in 0.665 ml of Hbuffer (100 mM HEPES, 150 mM NaCl, 2 mM MgCl₂, 1% BSA, pH 7.0) andpermeabilized by vortexing for 1 min after the addition of 0.055 ml eachof CHCl₃ and 0.1% SDS. The reaction was started by adding 0.125 ml of 40mM stock solution of CPRG (chlorophenylred-b-D-galactopyranoside) andincubation was continued at 30° C. until the color changed to red.Reactions were stopped by the addition of 0.4 ml of 1 M Na₂CO₃. Themixtures were microfuged for 5 min to remove cell debris and A₅₇₄ wasmeasured. β-galactosidase activity was expressed in Miller units.

Results

Isolation of ABE-binding protein factors—We employed a modified yeastone-hybrid system (Kim and Thomas, 1998; Kim et al., 1997) in order toisolate ABRE-binding factor(s) using the prototypical ABRE, Emla element(SEQ ID NO: 9; GGACACACGTGGCG). A cDNA expression library representing2×10⁷ cfu was constructed in a yeast expression vector pYESTrp2, using amixture of equal amounts of mRNAs isolated from ABA- and salt-treatedArabidopsis plants. The vector contains B42 activation domain (Ma andPtashne, 1987) whose expression is under the control of yeast GAL1promoter. Thus, expression of cDNAs, which are inserted as a fusion tothe activation domain, is inducible by galactose and repressed byglucose. The library DNA was used to transform a reporter yeast thatharbors the ABRE-containing HIS3 and lacZ reporters. From a screen of 4million yeast transformants, ca. 40 His⁺ blue colonies were obtained,among which 19 isolates were characterized further. Analysis of the cDNAinserts of the positive clones indicated that they could be divided into4 different groups according to their restriction patterns.Representative clones with longer inserts from each group were analyzedin more detail.

First, binding of the cDNA clones to the G/ABRE in yeast was confirmed.The G/ABRE-HIS3 reporter yeast was re-transformed with the libraryplasmid DNAs isolated from the representative clones. Growth pattern ofthe transformants on media lacking histidine was then examined tomeasure the HIS3 reporter activity. The result in FIG. 1 showed thattransformants obtained with all four clones could grow on a galactosemedium lacking histidine, but not on a glucose medium. In the sameassay, the transformed yeast containing a control reporter constructlacking the ABRE could not grow on the same galactose medium. Thus, theclones could activate HIS3 reporter gene reproducibly, indicating thatthey bind to the ABRE in yeast.

Next, nucleotide and deduced amino acid sequences of the representativeclones were determined. Clone 1, which represents two isolates,contained a cDNA insert of 1578 bp including a poly A(+) tail (FIG. 2A,SEQ ID NO. 1). An open reading frame (ORF) that is in frame with the B42domain was present within the sequence. The ORF, referred to as ABF1(ABRE-Binding Factor 1), contains an ATG initiation codon near theB42-cDNA junction, suggesting that it is a full-length clone. The aminoacid sequence starting from the initiation codon is shown in FIG. 2A(SEQ ID NO. 2). The insert of clone 2, which represents 8 isolates, is1654 bp long (FIG. 2B, SEQ ID NO. 3) and the longest ORF including aninitiation codon near the B42-cDNA junction encodes a protein of 416amino acids (ABF2) (FIG. 2B, SEQ ID NO. 4).

The insert of clone 11, representing 6 isolates, encoded a proteincontaining 434 amino acids. An ORF containing 366 amino acids was foundin clone 19 cDNA. The clones were partial, however, and the missing 5′portions were isolated using the available partial sequence informationon databases (Materials and methods). Sequencing of the full-lengthclones (FIGS. 2C and 2D, SEQ ID NOs. 5 and 7) showed that the originalclone 11 was missing the first 20 amino acids, and thus, full-lengthclone 11 encodes a protein containing 454 amino acids (ABF3) (FIG. 2C,SEQ ID NO. 6). The longest ORF of clone 19 is composed of 431 aminoacids (ABF4) (FIG. 2D, SEQ ID NO. 8).

ABFs are bZIP proteins—Analysis of the deduced amino acid sequence ofABF1 revealed that it has a basic region near its C-terminus (SEQ ID NO:2; FIGS. 2A and 3). The region immediately downstream of it contains 4heptad repeats of leucine, indicating that ABF1 is a bZIP protein(Landshulz et al., 1988). Similarly, other ABFs also have a basic regionfollowed by a leucine repeat region (SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8; FIGS. 2B-D and 3). The basic regions of ABF1 and ABF3 (SEQ ID NO:2, SEQ ID NO: 6, respectively) are identical to each other, and those ofABF 2 and (SEQ ID NO: 4, SEQ ID NO: 8, respectively) are also identical(FIG. 3). The two, shared basic regions are same except that one of thelysine residues of ABF1 and ABF3 is replaced by arginine in ABF2 andABF4. The analysis shows that a family of bZIP proteins with conservedbasic regions interacts with the G/ABRE.

ABFs also share several highly conserved regions outside the basicdomains. As shown in FIG. 3, the conserved regions are clustered in theN-terminal halves. Invariably, they contain one or two potentialphosphorylation sites. The N-most region, for example, contains onemultifunctional calmodulin-dependent protein kinase II (CaMK II) site(X-R-X-X-S*-X) (Kemp and Pearson, 1990) followed by a caseine kinase II(CKII) phosphorylation site (X-S/T*-X-X-D/E-X). One or two CaMK II or CKII phosphorylation sites are also present in other conserved regions.The middle portions of ABFs are highly variable and rich in glutaminecommonly found in transcriptional activation domains.

In vitro binding activity of ABFs—In order to test in vitro DNA-bindingactivity of ABFs, we performed electrophoretic mobility shift assay(EMSA) using recombinant ABF1 or ABF3 and probe DNAs containing a G/ orC/ABRE. Similar results were obtained with both proteins and the assayresult of ABF1 is shown in FIG. 4. A major shifted band was observed,with a weaker minor band (FIG. 4A, lane 2). Addition of excess,increasing amount of unlabeled probe DNA to the reaction mixture (lanes3 and 4) gradually abolished the binding, whereas the same amount of amutated oligonucleotide (lanes 5 and 6) did not. Thus, ABF1 and ABF3exhibited sequence-specific binding activity to the G/ABRE in vitro.

ABFs are similar to the Dc3 promoter-binding factors (DPBFs) (Kim andThomas, 1998; Kim et al., 1997) in their basic regions (see Discussion).Since DPBFs are known to interact not only with a G/ABRE but also withC/ABREs, we tested whether ABFs can interact with C/ABREs. To date, notranscriptional activators interacting with the element were reportedexcept the embryo-specific DPBFs. An oligonucleotide, hex-3 (SEQ ID NO:13; Lam and Chua, 1991), containing the C/ABRE core sequence (CGCGTG),was employed as a probe in an EMSA. As shown in FIG. 4B, a shifted bandwas observed (lane 2). The band formation was abolished by the additionof excess amounts of the cold probe DNA to the reaction mixture (lanes 3and 4). The competition was not observed with a mutated probe DNA (lanes5 and 6), demonstrating that the binding was specific to the C/ABRE.Thus, ABF1 and ABF3 could bind to a C/ABRE as well.

Binding site preference of ABF1—Our in vitro binding assay indicatedthat ABF1 and ABF3 can interact with both G/ and C/ABREs, althoughmutual competition assay (not shown) showed that they have higheraffinity to the G/ABRE. In order to investigate ABF binding sitesfurther, we performed a random binding site selection assay (RBSA)(Pollock and Treisman, 1990) (Materials and methods), using therecombinant ABF1. Shifted bands were visible on a mobility shift assaygel after three rounds of selection (FIG. 5A, top panel). Afterconfirming the binding of ABF1 to the probe DNAs from the final round ofselection (FIG. 5A, bottom panel) the DNA were cloned and sequenced.

The 44 selected sequences are presented in FIG. 5B. The sequences couldbe divided into 4 groups (groups IA, IAA, IB, and II) according to theirconsensus sequences. All of the group I sequences, except one (sequenceno. 49), contain an ACGT element, while the group II sequences containthe C/ABRE core. The most frequently selected sequences (30 of 44) arethose sharing a strong G/ABRE, CACGTGGC (Busk and Pages, 1998):gACACGTGGC (SEQ ID NO: 22; group IA) or CCACGTGGC (group IAA). The groupIAA element is similar to the prototypical ABRE, Em1a (SEQ ID NO: 9;GGACACGTGGC), while the group IAA consensus is the same as thepalindromic G/ABREs present in many ABA-inducible genes such as maizerab28, Arabidopsis kin1, cor6.6 and Adh1 genes (reviewed in Thomas et al1997). In some of the group IA sequences (sequence no. 38, 45 and 42),the GGC following the ACGT core is replaced by GTC, forming anotherpalindromic consensus sequence GACACGTGTC (SEQ ID NO: 23). The group IBsequences share a GNTGACGTGGC (SEQ ID NO: 24) consensus or its variantsdiffering in one or two bases flanking the ACGT core. Although theconserved element differs from those of group IA and IAA in the basespreceding the ACGT core, it contains the same ACGTG(G/t)C. Hence, thepreferred binding sites of ABF1 can be represented as ACGTG(G/t)C, withAC, CC or TG preceding it.

One of the selected sequences (no.24 of group II) contains the C/ABREcore sequence (CGCGTG). The three other group II sequences also containthe C/ABRE core. The element in them, however, is flanked by one of thegroup I consensus sequences, and thus, they contain both types of ABREs.Another sequence (no. 49 of group IB) does not contains the ACGT core;the C of the ACGT is replaced by A. The resulting AAGTGGA sequence issimilar to the half G-box (CCAAGTGG) of Arabidopsis Adh1 promoter, whichis required for high level ABA-induction of the gene (de Bruxelles etal., 1996). Thus, ABF1 interacts with sequences without the ACGT core,which includes the C/ABRE. The low selection frequency, however,suggests that ABF1's affinity to them is lower.

Expression of ABFs is ABA-inducible—Since we are interested inABA-inducible stress responsive factors, we investigatedABA-inducibility of ABF expression, by RNA gel blot analysis (FIG. 6A).With the ABF1 probe, no hybridization signal was detectable with RNAisolated from untreated plants, while a clear signal was detected withRNA from ABA-treated plants. Similar results were obtained with otherABF probes; while hybridization signals were weak (ABF2 and 4) orundetectable (ABF-3) with the RNA from untreated plants, distinctsignals were observed with the RNA sample from ABA-treated plants. Thus,expression of ABFs is ABA-inducible.

Although all are induced by ABA, the time course of ABA-inducedexpression of ABFs was not identical to each other (FIG. 6B). ABF1 RNAlevel reached a peak approximately 2 hours after ABA treatment started,remained same up to 12 hours and decreased to the uninduced level after16 hours. ABF2 and ABF4 expression appeared to be induced faster,reaching a plateau after 30 min of ABA treatment. Afterwards, their RNAlevel remained relatively same until 24 hour. The induction pattern ofABF3 was similar to those of ABF2 and ABF4 except that it reached thepeak level later, i.e., after 2 hours.

We also examined the effect of various environmental stresses on theexpression of ABFs. The results (FIG. 6A) showed that expression of ABF1was induced by cold treatment, but not by other stress treatments. Withthe same RNA samples, ABF2 and ABF3, on the other hand, were not inducedby cold, but by high salt treatment. ABF4 expression was induced by allthree treatments, although induction level after cold treatment wasrelatively low. Expression of ABFs is, thus, inducible also by variousenvironmental stresses and their induction patterns are differential,suggesting that they function in different stress responsive pathways.

ABFs can transactivate an ABRE-containing reporter gene in yeast—Ourresult so far demonstrated that ABF1, and probably other ABFs too, canbind to various ABREs and that their expression is both ABA- andstress-dependent. Thus, ABFs have potential to activate a large numberof ABA/stress responsive genes, if they have transactivation capability.We therefore investigated whether ABFs can activate an ABRE-containingreporter gene. Coding regions of ABFs were cloned into a yeastexpression vector and the constructs were individually introduced into ayeast strain that harbored a G/ABRE-containing lacZ reporter geneintegrated into the chromosome. Subsequently, reporter enzyme activitywas measured.

With the ABF1 construct, β-galactosidase activity was 6 times higherthan that obtained with the control construct (FIG. 7, top panel). Noenzyme activity was detectable with the same ABF1 construct when areporter lacking the ABRE was used. Thus, ABF1 can transactivate thereporter gene and the activation is ABRE-dependent. With the ABF2construct, reporter enzyme activity two times higher than the backgroundactivity was detected, indicating that the factor also can transactivatethe reporter gene (FIG. 7, top panel). Likewise, ABF3 and 4 couldtransactivate the reporter gene (FIG. 7, bottom panel). The activationlevel of ABF3 was higher than the ABF1's, while ABF4 showed weakeractivation. The result of our transactivation assay demonstrates thatABFs can activate an ABRE-containing gene in yeast.

Discussion

Numerous studies, both genetic and biochemical, show that ABA mediatesstress response in vegetative tissues, although not all stress responsesare ABA-dependent (Leung and Giraudat, 1998; Shinozaki andYamaguchi-Shinozaki, 1996; Thomashow, 1998; Ingram and Bartels, 1996).Central to the response is the ABA-regulation of gene expression throughG/ABREs or C/ABREs. Transcription factors mediating ABA-independent coldand drought responses have been reported recently (Jaglo-Ottosen et al.,1998; Liu et al., 1998). However, those regulating ABA-dependent stressresponse via the G/ or the C/ABREs have yet to be identified. Among theABRE-binding factors mentioned earlier, TAF-1 is known not to bedirectly involved in ABA responsive gene expression (Oeda et al., 1991),while EmBP-1 and DPBFs are highly embryo-specific (Kim and Thomas, 1998;Hollung et al., 1997). GBF3 and a homology-based cloned factor OSBZ8,although inducible by ABA, are from cultured cells or from embryos (Luet al., 1996; Nakagawa et al., 1996). Taken together with the lack ofdata demonstrating their role in ABA or stress response, it is likelythat unknown factors may mediate ABA-responsive gene expression invegetative tissues.

In a search for such transcription factors, we isolated a family ofG/ABRE-binding proteins from young Arabidopsis plants treated with ABAor high salt. The factors, referred to as ABFs, are ABA/stress-induciblebZIP class transcription factors with shared basic regions. Sequencecomparison with known ABRE-binding factors indicated that, although theydo not show any significant homology to other factors, they are similarto the Dc3 promoter-binding factors (DPBFs) (Kim and Thomas, 1998; Kimet al., 1997). DPBFs have been isolated from a seed-specific librarybased on their interaction with a lea gene promoter containing both G/and C/ABREs. The two family members are nearly identical in their basicregions (FIG. 8A), and their DNA-binding properties are similar in thatthey can interact with both types of ABREs. Some of the conservedphosphorylation sites within the N-terminal halves of ABFs are alsoconserved in DPBFs. However, ABFs diverge from the DPBFs outside thebasic regions and their immediate flanking sequences, overall identitybeing in the range of 30-40%. As a result, they form a subfamilydistinct from DPBFs and also from other known factors, as shown in FIG.8B. Furthermore, their expression patterns are different from those ofDPBFs; i.e., DPBFs' expression is embryo-specific. Cloning of ABFs showsthat two related subfamilies of ABRE-binding factors are present in seedand in vegetative tissues, respectively. The presence of distinctfactors in the tissues that have similar ABRE-binding affinity has beendemonstrated in maize (Pla et al., 1993).

ABFs contain regions highly conserved among them apart from the basicregions. Thus, ABFs appear to share some properties other thanDNA-binding activity. The conserved regions, however, do not have anyeasily recognizable motifs except that two of them can form α-helix, andthus, their function remains to be identified. They may be involved innuclear translocation, DNA-binding, transcriptional activation, orinteraction with other regulatory proteins. Whatever their function maybe, the conservation of potential phosphorylation sites within theregions suggests that it is probably modulated by post-translationalmodification.

Our in vitro binding assay showed that the most preferred binding siteof ABF1 in vitro can be represented as CACGTGGC (FIG. 5B). The element,first identified as EmBP-1 recognition site (Guiltinan et al., 1990),are highly conserved among ABA/stress inducible promoters and stronglyaffect ABA-inducibility in vivo (Busk and Pages, 1998). Together withthe fact that ABF1 is ABA/stress-inducible and has transcriptionalactivity, this suggests that ABF1 can potentially activate a largenumber of ABA/stress responsive genes. Also, ABF1 can bind to otherABREs including the C/ABREs, further supporting the broad spectrum ofpotential ABF1 target genes. The affinity to C/ABREs, however, wasrelatively low in vitro.

The expression pattern of ABFs suggests that each ABF is likely to beinvolved in different stress signaling pathways. Although all areABA-inducible and can bind to same ABREs, they are differentiallyregulated by various environmental stresses. ABF1 expression is inducedby cold, ABF 2 and ABF3 by high salt, and ABF4 by cold, high salt anddrought. The simplest interpretation of the result would be that ABF1 isinvolved in cold signal transduction, while ABF2 and ABF3 function inosmotic stress signaling. ABF4, on the other hand, appears toparticipate in multiple stress responses. In addition, ABFs differ intheir ABA induction patterns. Expression of ABF1 was induced ratherslowly (FIG. 6B) and the accumulation of its RNA was transient, whileinduction of other ABFs appeared faster and their RNA levels remainedrelatively stable once reached a plateau. The multiplicity ofABA-dependent stress signaling pathways has been demonstrated inArabidopsis by genetic analysis (Leung and Giraudat, 1998; Ishitani etal., 1997). Our result suggests further that multiple transcriptionfactors are likely to function in these signal transduction cascadesthrough common ABREs.

ABA-dependent stress responsive gene expression is critical to plantgrowth and productivity. Here, we reported a family of transcriptionfactors that interact with cis-regulatory elements mediating thisprocess. Although their specific roles in planta remains to bedetermined, our data presented here suggest that they are likely to beinvolved in various ABA-mediated stress responses. They can bind toABREs highly conserved among stress responsive promoters. They cantransactivate an ABRE-containing reporter gene. Their expression isinduced by ABA and by various environmental stresses. Hence, ABFs areexcellent targets of genetic manipulation for the generation of stresstolerant transgenic plants.

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83 1 1578 DNA Arabidopsis thaliana 1 aaagggtctg attcgtttgt tttttcactgaagaatttgg aaggaagtga ttccgttgtg 60 aaacagaaaa gaagtatggg tactcacattgatatcaaca acttaggcgg cgatacttct 120 agagggaatg agtcaaagcc attggcgaggcagtcttcgt tatattcctt aacgtttgat 180 gagcttcaga gcacattagg tgagccggggaaagattttg ggtctatgaa tatggatgag 240 ttactcaaga acatatggac tgctgaggatactcaagcct ttatgactac tacatcttcg 300 gttgcagccc cgggacctag tggttttgttccgggaggaa atggtttaca gaggcaaggc 360 tccttgacct tgcctagaac gcttagtcagaagactgtcg atgaagtctg gaaatacctg 420 aattcgaaag aaggtagtaa tgggaatactggaacggatg cgcttgagag gcaacagact 480 ttaggggaaa tgactctgga agatttcttactccgtgctg gcgttgttaa agaagataat 540 actcagcaga acgaaaacag tagtagcgggttttatgcta acaacggtgc tgctggtttg 600 gagtttggat ttggtcagcc gaatcaaaacagcatatcgt tcaacgggaa caatagttct 660 atgatcatga atcaagcacc tggtttaggcctcaaagttg gtggaaccat gcagcagcag 720 cagcagccac atcagcagca gttgcagcagccacatcaga gactgcctcc aactatcttt 780 ccaaaacaag cgaatgtaac atttgcggcgcctgtaaata tggtcaacag gggtttattt 840 gagactagcg cagatggtcc agccaacagtaatatgggag gagcaggggg tactgttaca 900 gctacttctc ctgggacgag cagtgcagaaaacaatactt ggtcatcacc agttccttac 960 gtgtttggtc ggggaagaag aagcaatacgggcctggaga aggttgttga gagaaggcaa 1020 aagagaatga tcaagaatcg ggaatccgctgctagatcaa gggctcgaaa acaggcttat 1080 accttggaac tggaagctga gattgaaagtctcaagctag tgaatcaaga tttgcagaag 1140 aaacaggctg aaataatgaa aacccataatagtgagctaa aggaattttc gaagcagcct 1200 ccattgctgg ccaaaagaca atgcttgagaagaaccctta ccggtccgtg gtaagaaggt 1260 gaagtcaaag caagaagaac ctgctaatgtaatacaggac cactcaaaag gaagacactg 1320 ggagagtaat atgtaataga agatagtgctactgtacagg agaaattaca gagacgctta 1380 caatgtagaa atcttttgag ctgaatttaactaagagtgc agtctgtgta gagtatgaga 1440 gctttcaata tgaattcata attttcataaacatatgtaa aactttcaga tttagctata 1500 gagaagatgt gactaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560 aaaaaaaaaa aaaaaaaa 1578 2 392 PRTArabidopsis thaliana 2 Met Gly Thr His Ile Asp Ile Asn Asn Leu Gly GlyAsp Thr Ser Arg 1 5 10 15 Gly Asn Glu Ser Lys Pro Leu Ala Arg Gln SerSer Leu Tyr Ser Leu 20 25 30 Thr Phe Asp Glu Leu Gln Ser Thr Leu Gly GluPro Gly Lys Asp Phe 35 40 45 Gly Ser Met Asn Met Asp Glu Leu Leu Lys AsnIle Trp Thr Ala Glu 50 55 60 Asp Thr Gln Ala Phe Met Thr Thr Thr Ser SerVal Ala Ala Pro Gly 65 70 75 80 Pro Ser Gly Phe Val Pro Gly Gly Asn GlyLeu Gln Arg Gln Gly Ser 85 90 95 Leu Thr Leu Pro Arg Thr Leu Ser Gln LysThr Val Asp Glu Val Trp 100 105 110 Lys Tyr Leu Asn Ser Lys Glu Gly SerAsn Gly Asn Thr Gly Thr Asp 115 120 125 Ala Leu Glu Arg Gln Gln Thr LeuGly Glu Met Thr Leu Glu Asp Phe 130 135 140 Leu Leu Arg Ala Gly Val ValLys Glu Asp Asn Thr Gln Gln Asn Glu 145 150 155 160 Asn Ser Ser Ser GlyPhe Tyr Ala Asn Asn Gly Ala Ala Gly Leu Glu 165 170 175 Phe Gly Phe GlyGln Pro Asn Gln Asn Ser Ile Ser Phe Asn Gly Asn 180 185 190 Asn Ser SerMet Ile Met Asn Gln Ala Pro Gly Leu Gly Leu Lys Val 195 200 205 Gly GlyThr Met Gln Gln Gln Gln Gln Pro His Gln Gln Gln Leu Gln 210 215 220 GlnPro His Gln Arg Leu Pro Pro Thr Ile Phe Pro Lys Gln Ala Asn 225 230 235240 Val Thr Phe Ala Ala Pro Val Asn Met Val Asn Arg Gly Leu Phe Glu 245250 255 Thr Ser Ala Asp Gly Pro Ala Asn Ser Asn Met Gly Gly Ala Gly Gly260 265 270 Thr Val Thr Ala Thr Ser Pro Gly Thr Ser Ser Ala Glu Asn AsnThr 275 280 285 Trp Ser Ser Pro Val Pro Tyr Val Phe Gly Arg Gly Arg ArgSer Asn 290 295 300 Thr Gly Leu Glu Lys Val Val Glu Arg Arg Gln Lys ArgMet Ile Lys 305 310 315 320 Asn Arg Glu Ser Ala Ala Arg Ser Arg Ala ArgLys Gln Ala Tyr Thr 325 330 335 Leu Glu Leu Glu Ala Glu Ile Glu Ser LeuLys Leu Val Asn Gln Asp 340 345 350 Leu Gln Lys Lys Gln Ala Glu Ile MetLys Thr His Asn Ser Glu Leu 355 360 365 Lys Glu Phe Ser Lys Gln Pro ProLeu Leu Ala Lys Arg Gln Cys Leu 370 375 380 Arg Arg Thr Leu Thr Gly ProTrp 385 390 3 1654 DNA Arabidopsis thaliana 3 cccaaacgaa gaaccaaacattttgaaatt ttttgggaaa attacaaagc acacgaattt 60 agcaaaaaga tccagttattaggtggaagc agattttgta gaaaaatgga tggtagtatg 120 aatttgggga atgagccaccaggagatggt ggtggaggtg gagggttgac tagacaaggt 180 tcgatatact cgttgacgtttgatgagttt cagagcagtg tagggaaaga ttttgggtca 240 atgaacatgg atgagttgttaaagaatata tggagtgctg aagaaacaca agccatggct 300 agtggtgtgg ttccagttcttggtggaggt caagagggtt tgcagctgca gaggcaaggc 360 tcgttgactc tgcctcgaacgcttagtcag aagacggttg atcaagtttg gaaagatcta 420 tccaaagttg gaagtagtggagtaggggga agtaacttgt ctcaggtggc tcaggctcag 480 agtcagagtc agagtcagaggcagcaaaca ttaggtgaag taactttgga ggagtttttg 540 gttcgtgctg gtgttgtgagagaggaagct caggttgctg caagagctca gattgctgag 600 aacaataaag gcggttactttggtaatgat gccaacacag gtttctctgt cgagtttcag 660 cagccttctc cacgagttgttgccgctggt gtaatgggaa atcttggtgc agagactgca 720 aattctttgc aggttcaaggttctagtttg cctctgaatg tgaatggagc tagaacaaca 780 taccagcaat cgcaacagcaacagccaatc atgcctaagc agcctggttt tggttatgga 840 acacaaatgg gtcagcttaatagtcctggg ataagaggtg gtggtcttgt gggacttgga 900 gatcagtctt taacgaacaatgtgggcttt gtccaaggtg cttctgctgc aattcctgga 960 gctttaggcg ttggtgctgtgtcgcctgtt acgccattgt catcagaagg gatagggaag 1020 agtaatggtg attcttcatcactctctccg tctccttaca tgtttaatgg tggtgtgaga 1080 ggtagaaaga gtggcactgtggagaaagtt gtagagagaa ggcaaaggag aatgataaag 1140 aaccgagaat cagctgcaaggtcccgggcc aggaaacagg cttacaccgt ggagcttgaa 1200 gctgaagttg caaagttaaaggaagagaat gacgagttac aacgaaagca ggcaaggatc 1260 atggaaatgc aaaagaatcaggagacggag atgaggaatc ttctgcaagg aggtccaaag 1320 aaaaagctga ggaggacagagtcgggacct tggtgaatca atcaatgcca tcatacttag 1380 tttctgtaga taaatgacatcccacttagg tgttttagtt gaattagact taatagagaa 1440 gagctttcat cgtttatattgtaagctctc tccatatatg ttatgttttt tacatacaca 1500 ggatcatcag aatctcttttgctttattta gaccaagaat tttgtgtgtg tttctcgttg 1560 ttgtttgtcg ttgtcgctattaaacctcaa aatgtacttt cttgatcttg gagttaccaa 1620 ttttgaagaa ttgaagtgttgtttggttaa aaaa 1654 4 416 PRT Arabidopsis thaliana 4 Met Asp Gly SerMet Asn Leu Gly Asn Glu Pro Pro Gly Asp Gly Gly 1 5 10 15 Gly Gly GlyGly Leu Thr Arg Gln Gly Ser Ile Tyr Ser Leu Thr Phe 20 25 30 Asp Glu PheGln Ser Ser Val Gly Lys Asp Phe Gly Ser Met Asn Met 35 40 45 Asp Glu LeuLeu Lys Asn Ile Trp Ser Ala Glu Glu Thr Gln Ala Met 50 55 60 Ala Ser GlyVal Val Pro Val Leu Gly Gly Gly Gln Glu Gly Leu Gln 65 70 75 80 Leu GlnArg Gln Gly Ser Leu Thr Leu Pro Arg Thr Leu Ser Gln Lys 85 90 95 Thr ValAsp Gln Val Trp Lys Asp Leu Ser Lys Val Gly Ser Ser Gly 100 105 110 ValGly Gly Ser Asn Leu Ser Gln Val Ala Gln Ala Gln Ser Gln Ser 115 120 125Gln Ser Gln Arg Gln Gln Thr Leu Gly Glu Val Thr Leu Glu Glu Phe 130 135140 Leu Val Arg Ala Gly Val Val Arg Glu Glu Ala Gln Val Ala Ala Arg 145150 155 160 Ala Gln Ile Ala Glu Asn Asn Lys Gly Gly Tyr Phe Gly Asn AspAla 165 170 175 Asn Thr Gly Phe Ser Val Glu Phe Gln Gln Pro Ser Pro ArgVal Val 180 185 190 Ala Ala Gly Val Met Gly Asn Leu Gly Ala Glu Thr AlaAsn Ser Leu 195 200 205 Gln Val Gln Gly Ser Ser Leu Pro Leu Asn Val AsnGly Ala Arg Thr 210 215 220 Thr Tyr Gln Gln Ser Gln Gln Gln Gln Pro IleMet Pro Lys Gln Pro 225 230 235 240 Gly Phe Gly Tyr Gly Thr Gln Met GlyGln Leu Asn Ser Pro Gly Ile 245 250 255 Arg Gly Gly Gly Leu Val Gly LeuGly Asp Gln Ser Leu Thr Asn Asn 260 265 270 Val Gly Phe Val Gln Gly AlaSer Ala Ala Ile Pro Gly Ala Leu Gly 275 280 285 Val Gly Ala Val Ser ProVal Thr Pro Leu Ser Ser Glu Gly Ile Gly 290 295 300 Lys Ser Asn Gly AspSer Ser Ser Leu Ser Pro Ser Pro Tyr Met Phe 305 310 315 320 Asn Gly GlyVal Arg Gly Arg Lys Ser Gly Thr Val Glu Lys Val Val 325 330 335 Glu ArgArg Gln Arg Arg Met Ile Lys Asn Arg Glu Ser Ala Ala Arg 340 345 350 SerArg Ala Arg Lys Gln Ala Tyr Thr Val Glu Leu Glu Ala Glu Val 355 360 365Ala Lys Leu Lys Glu Glu Asn Asp Glu Leu Gln Arg Lys Gln Ala Arg 370 375380 Ile Met Glu Met Gln Lys Asn Gln Glu Thr Glu Met Arg Asn Leu Leu 385390 395 400 Gln Gly Gly Pro Lys Lys Lys Leu Arg Arg Thr Glu Ser Gly ProTrp 405 410 415 5 1685 DNA Arabidopsis thaliana 5 gaagcttgat cctcctagttgtacgaaagc ttgagtaatg gggtctagat taaacttcaa 60 gagctttgtt gatggtgtgagtgagcagca gccaacggtg gggactagtc ttccattgac 120 taggcagaac tctgtgttctcgttaacctt tgatgagttt cagaactcat ggggtggtgg 180 aattgggaaa gattttgggtctatgaacat ggatgagctc ttgaagaaca tttggactgc 240 agaggaaagt cattcaatgatgggaaacaa taccagttac accaacatca gcaatggtaa 300 tagtggaaac actgttattaacggcggtgg taacaacatt ggtgggttag ctgttggtgt 360 gggaggagaa agtggtggttttttcactgg tgggagtttg cagagacaag gttcacttac 420 cttgcctcgg acgattagtcagaaaagggt tgatgatgtc tggaaggagc tgatgaagga 480 ggatgacatt ggaaatggtgttgttaatgg tgggacaagc ggaattccgc agaggcaaca 540 aacgctggga gagatgactttggaggagtt tttggtcagg gctggtgtgg ttagggaaga 600 acctcaaccg gtggagagtgtaactaactt caatggcgga ttctatggat ttggcagtaa 660 tggaggtctt gggacagctagtaatgggtt tgttgcaaac caacctcaag atttgtcagg 720 aaatggagta gcggtgagacaggatctgct gactgctcaa actcagccac tacagatgca 780 gcagccacag atggtgcagcagccacagat ggtgcagcag ccgcaacaac tgatacagac 840 gcaggagagg ccttttcccaaacagaccac tatagcattt tccaacactg ttgatgtggt 900 taaccgttct caacctgcaacacagtgcca ggaagtgaag ccttcaatac ttggaattca 960 taaccatcct atgaacaacaatctactgca agctgtcgat tttaaaacag gagtaacggt 1020 tgcagcagta tctcctggaagccagatgtc acctgatctg actccaaaga gcgccctgga 1080 tgcatctttg tcccctgttccttacatgtt tgggcgagtg agaaaaacag gtgcagttct 1140 ggagaaagtg attgagagaaggcaaaaaag gatgataaag aatagggaat cagctgcaag 1200 atcccgcgct cgcaagcaagcttatacgat ggaactggaa gcagaaattg cgcaactcaa 1260 agaattgaat gaagagttgcagaagaaaca agttgaaatc atggaaaagc agaaaaatca 1320 gcttctggag cctctgcgccagccatgggg aatgggatgc aaaaggcaat gcttgcgaag 1380 gacattgacg ggtccctggtagagcttata atggcgtcta aggaacccaa caaagcgccg 1440 aagttataga acaactcagaagatagaaag ctagctttgt acgtagttta ggcaggttct 1500 gtgggtgatt gtaaatcttgaagtgtggcg gatttgacag agatagataa acacatatct 1560 gttctatttt cctaaatcttttggttttat cttcctgatg taatggatct ttatcatttg 1620 tcttgaacat ctttgtgacttaaccagagt gaatttatct tgtatctaaa aaaaaaaaaa 1680 aaaaa 1685 6 454 PRTArabidopsis thaliana 6 Met Gly Ser Arg Leu Asn Phe Lys Ser Phe Val AspGly Val Ser Glu 1 5 10 15 Gln Gln Pro Thr Val Gly Thr Ser Leu Pro LeuThr Arg Gln Asn Ser 20 25 30 Val Phe Ser Leu Thr Phe Asp Glu Phe Gln AsnSer Trp Gly Gly Gly 35 40 45 Ile Gly Lys Asp Phe Gly Ser Met Asn Met AspGlu Leu Leu Lys Asn 50 55 60 Ile Trp Thr Ala Glu Glu Ser His Ser Met MetGly Asn Asn Thr Ser 65 70 75 80 Tyr Thr Asn Ile Ser Asn Gly Asn Ser GlyAsn Thr Val Ile Asn Gly 85 90 95 Gly Gly Asn Asn Ile Gly Gly Leu Ala ValGly Val Gly Gly Glu Ser 100 105 110 Gly Gly Phe Phe Thr Gly Gly Ser LeuGln Arg Gln Gly Ser Leu Thr 115 120 125 Leu Pro Arg Thr Ile Ser Gln LysArg Val Asp Asp Val Trp Lys Glu 130 135 140 Leu Met Lys Glu Asp Asp IleGly Asn Gly Val Val Asn Gly Gly Thr 145 150 155 160 Ser Gly Ile Pro GlnArg Gln Gln Thr Leu Gly Glu Met Thr Leu Glu 165 170 175 Glu Phe Leu ValArg Ala Gly Val Val Arg Glu Glu Pro Gln Pro Val 180 185 190 Glu Ser ValThr Asn Phe Asn Gly Gly Phe Tyr Gly Phe Gly Ser Asn 195 200 205 Gly GlyLeu Gly Thr Ala Ser Asn Gly Phe Val Ala Asn Gln Pro Gln 210 215 220 AspLeu Ser Gly Asn Gly Val Ala Val Arg Gln Asp Leu Leu Thr Ala 225 230 235240 Gln Thr Gln Pro Leu Gln Met Gln Gln Pro Gln Met Val Gln Gln Pro 245250 255 Gln Met Val Gln Gln Pro Gln Gln Leu Ile Gln Thr Gln Glu Arg Pro260 265 270 Phe Pro Lys Gln Thr Thr Ile Ala Phe Ser Asn Thr Val Asp ValVal 275 280 285 Asn Arg Ser Gln Pro Ala Thr Gln Cys Gln Glu Val Lys ProSer Ile 290 295 300 Leu Gly Ile His Asn His Pro Met Asn Asn Asn Leu LeuGln Ala Val 305 310 315 320 Asp Phe Lys Thr Gly Val Thr Val Ala Ala ValSer Pro Gly Ser Gln 325 330 335 Met Ser Pro Asp Leu Thr Pro Lys Ser AlaLeu Asp Ala Ser Leu Ser 340 345 350 Pro Val Pro Tyr Met Phe Gly Arg ValArg Lys Thr Gly Ala Val Leu 355 360 365 Glu Lys Val Ile Glu Arg Arg GlnLys Arg Met Ile Lys Asn Arg Glu 370 375 380 Ser Ala Ala Arg Ser Arg AlaArg Lys Gln Ala Tyr Thr Met Glu Leu 385 390 395 400 Glu Ala Glu Ile AlaGln Leu Lys Glu Leu Asn Glu Glu Leu Gln Lys 405 410 415 Lys Gln Val GluIle Met Glu Lys Gln Lys Asn Gln Leu Leu Glu Pro 420 425 430 Leu Arg GlnPro Trp Gly Met Gly Cys Lys Arg Gln Cys Leu Arg Arg 435 440 445 Thr LeuThr Gly Pro Trp 450 7 1737 DNA Arabidopsis thaliana 7 gaacaagggttttagggctt ggatgctttg ttttcattga aaaagaagta gaaggagtgt 60 atacaaggattatgggaact cacatcaatt tcaacaactt aggaggtggt ggtcatcctg 120 gaggggaagggagtagtaac cagatgaagc caacgggtag tgtcatgccc ttggctaggc 180 agtcctcggtctactccctt acctttgatg agttacagaa cacactaggt ggaccgggaa 240 aagatttcgggtcgatgaac atggatgaac tcctgaagag catatggact gctgaggaag 300 ctcaggccatggccatgact tctgcgccag ctgctacagc ggtagcgcaa cctggtgctg 360 gtatcccacccccaggtggg aatctccaga ggcaaggttc gttgacgttg cctagaacaa 420 ttagtcagaagactgttgat gaggtgtgga aatgtttgat caccaaggat ggtaatatgg 480 aaggtagcagcggaggcggt ggtgagtcga atgtgcctcc tggaaggcaa cagactttag 540 gggaaatgacacttgaagaa tttctgttcc gtgctggggt tgtaagagaa gataactgtg 600 ttcaacagatgggtcaggtc aacggaaaca ataacaatgg gttttatggt aacagcactg 660 ctgctggcggcttaggtttt ggatttggtc agccaaatca aaacagcata acattcaatg 720 gtactaatgattctatgatc ttgaatcagc cacctggttt agggctcaaa atgggtggaa 780 caatgcagcagcaacaacaa caacagcagt tgcttcagca gcaacaacag cagatgcagc 840 agctgaatcagcctcatcca cagcagcggc tgcctcaaac catttttcct aaacaagcaa 900 acgtagcattttctgcgcct gtgaatataa ccaacaaggg ttttgctggg gctgcaaata 960 attctatcaacaataataat ggattagcta gttacggagg aaccggggtc actgttgcag 1020 caacttctccaggaacaagc agcgcagaaa ataattcttt atcaccagtt ccgtatgtgc 1080 ttaatcgaggacgaagaagc aatacaggtc tagagaaggt tatcgagagg aggcaaagga 1140 gaatgatcaagaatcgggaa tcagctgcta gatcaagagc tcgaaagcag gcttatacat 1200 tggaactggaagccgaaatt gaaaagctca agaaaacgaa tcaagaactg cagaaaaaac 1260 aggctgaaatggtggaaatg cagaagaatg agctgaaaga aacgtcgaag cgaccgtggg 1320 gcagcaaaaggcaatgcttg agaaggacat taaccggacc atggtgaagg atgaagcaac 1380 aagaacggatgaaccagact cctagcttgg gattaatgta ataggatagt gctacctgta 1440 caggagattaagagaaattg agtgaaagat ctaggttaca gagtaggaga gagttttcat 1500 tatgaataaatgacattttg tgccctgacc tttgttagtt taggtttaga ttatcctctg 1560 ttattgacttattgtgcttt ctggttgtta gggtttctaa aagacatagt tgtttatata 1620 tatgtctgactttgtattcc ggatttggtt ctcttgtgtc attaacttgg gtttagccat 1680 tattacttaagagtggcaac gaaatcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1737 8 431 PRTArabidopsis thaliana 8 Met Gly Thr His Ile Asn Phe Asn Asn Leu Gly GlyGly Gly His Pro 1 5 10 15 Gly Gly Glu Gly Ser Ser Asn Gln Met Lys ProThr Gly Ser Val Met 20 25 30 Pro Leu Ala Arg Gln Ser Ser Val Tyr Ser LeuThr Phe Asp Glu Leu 35 40 45 Gln Asn Thr Leu Gly Gly Pro Gly Lys Asp PheGly Ser Met Asn Met 50 55 60 Asp Glu Leu Leu Lys Ser Ile Trp Thr Ala GluGlu Ala Gln Ala Met 65 70 75 80 Ala Met Thr Ser Ala Pro Ala Ala Thr AlaVal Ala Gln Pro Gly Ala 85 90 95 Gly Ile Pro Pro Pro Gly Gly Asn Leu GlnArg Gln Gly Ser Leu Thr 100 105 110 Leu Pro Arg Thr Ile Ser Gln Lys ThrVal Asp Glu Val Trp Lys Cys 115 120 125 Leu Ile Thr Lys Asp Gly Asn MetGlu Gly Ser Ser Gly Gly Gly Gly 130 135 140 Glu Ser Asn Val Pro Pro GlyArg Gln Gln Thr Leu Gly Glu Met Thr 145 150 155 160 Leu Glu Glu Phe LeuPhe Arg Ala Gly Val Val Arg Glu Asp Asn Cys 165 170 175 Val Gln Gln MetGly Gln Val Asn Gly Asn Asn Asn Asn Gly Phe Tyr 180 185 190 Gly Asn SerThr Ala Ala Gly Gly Leu Gly Phe Gly Phe Gly Gln Pro 195 200 205 Asn GlnAsn Ser Ile Thr Phe Asn Gly Thr Asn Asp Ser Met Ile Leu 210 215 220 AsnGln Pro Pro Gly Leu Gly Leu Lys Met Gly Gly Thr Met Gln Gln 225 230 235240 Gln Gln Gln Gln Gln Gln Leu Leu Gln Gln Gln Gln Gln Gln Met Gln 245250 255 Gln Leu Asn Gln Pro His Pro Gln Gln Arg Leu Pro Gln Thr Ile Phe260 265 270 Pro Lys Gln Ala Asn Val Ala Phe Ser Ala Pro Val Asn Ile ThrAsn 275 280 285 Lys Gly Phe Ala Gly Ala Ala Asn Asn Ser Ile Asn Asn AsnAsn Gly 290 295 300 Leu Ala Ser Tyr Gly Gly Thr Gly Val Thr Val Ala AlaThr Ser Pro 305 310 315 320 Gly Thr Ser Ser Ala Glu Asn Asn Ser Leu SerPro Val Pro Tyr Val 325 330 335 Leu Asn Arg Gly Arg Arg Ser Asn Thr GlyLeu Glu Lys Val Ile Glu 340 345 350 Arg Arg Gln Arg Arg Met Ile Lys AsnArg Glu Ser Ala Ala Arg Ser 355 360 365 Arg Ala Arg Lys Gln Ala Tyr ThrLeu Glu Leu Glu Ala Glu Ile Glu 370 375 380 Lys Leu Lys Lys Thr Asn GlnGlu Leu Gln Lys Lys Gln Ala Glu Met 385 390 395 400 Val Glu Met Gln LysAsn Glu Leu Lys Glu Thr Ser Lys Arg Pro Trp 405 410 415 Gly Ser Lys ArgGln Cys Leu Arg Arg Thr Leu Thr Gly Pro Trp 420 425 430 9 12 DNAArabidopsis thaliana 9 ggacacgtgg cg 12 10 36 DNA Arabidopsis thaliana10 ggacacgtgg cgggacacgt ggcgggacac gtggcg 36 11 24 DNA Arabidopsisthaliana 11 aattccggac acgtggcgta agct 24 12 24 DNA Arabidopsis thaliana12 aattccggac ctacagccta agct 24 13 24 DNA Arabidopsis thaliana 13aattccggac gcgtggccta agct 24 14 24 DNA Arabidopsis thaliana 14aattccggac ctacagccta agct 24 15 24 DNA Arabidopsis thaliana 15gaagcttgat cctcctagtt gtac 24 16 22 DNA Arabidopsis thaliana 16atttgaacaa gggttttagg gc 22 17 24 DNA Arabidopsis thaliana 17 ttacaatcacccacagaacc tgcc 24 18 21 DNA Arabidopsis thaliana 18 gatttcgttgccactcttaa g 21 19 41 DNA Arabidopsis thaliana misc_feature (1)...(41) n= A,T,C or G 19 cagttgagcc gatcctgtcg nsgaggcgaa tcagtgcaac t 41 20 20DNA Arabidopsis thaliana 20 agttgcactg aattcgcctc 20 21 20 DNAArabidopsis thaliana 21 cagttgagcg gatcctgtcg 20 22 10 DNA Arabidopsisthaliana 22 gacacgtgtc 10 23 10 DNA Arabidopsis thaliana 23 gacacgtgtc10 24 11 DNA Arabidopsis thaliana misc_feature (1)...(11) n = A,T,C or G24 gntgacgtgg c 11 25 61 PRT Arabidopsis thaliana 25 Pro Val Glu Lys ValVal Glu Arg Arg Gln Arg Arg Met Ile Lys Asn 1 5 10 15 Arg Glu Ser AlaAla Arg Ser Arg Ala Arg Lys Gln Ala Tyr Thr Val 20 25 30 Glu Leu Glu AlaGlu Leu Asn Met Leu Lys Glu Glu Asn Ala Gln Leu 35 40 45 Lys Gln Ala LeuAla Glu Ile Glu Arg Lys Arg Lys Gln 50 55 60 26 61 PRT Arabidopsisthaliana 26 Pro Met Glu Lys Thr Val Glu Arg Arg Gln Lys Arg Met Ile LysAsn 1 5 10 15 Arg Glu Ser Ala Ala Arg Ser Arg Ala Arg Lys Gln Ala TyrThr His 20 25 30 Glu Leu Glu Asn Lys Val Ser Arg Leu Glu Glu Glu Asn GluArg Leu 35 40 45 Arg Arg Glu Lys Glu Val Glu Lys Val Ile Pro Trp Val 5055 60 27 61 PRT Arabidopsis thaliana 27 Pro Ile Glu Lys Thr Val Glu ArgArg Gln Lys Arg Met Ile Lys Asn 1 5 10 15 Arg Glu Ser Ala Ala Arg SerArg Ala Arg Lys Gln Ala Tyr Thr His 20 25 30 Glu Leu Glu Asn Lys Ile SerArg Leu Glu Glu Glu Asn Glu Leu Leu 35 40 45 Lys Arg Gln Lys Glu Val GlyMet Val Leu Pro Ser Ala 50 55 60 28 61 PRT Arabidopsis thaliana 28 ProAsn Asp Thr Thr Asp Glu Arg Lys Arg Lys Arg Met Leu Ser Asn 1 5 10 15Arg Glu Ser Ala Arg Arg Ser Arg Ala Arg Lys Gln Gln Arg Leu Glu 20 25 30Glu Leu Val Ala Glu Val Ala Arg Leu Gln Ala Glu Asn Ala Ala Thr 35 40 45Gln Ala Arg Thr Ala Ala Leu Glu Arg Asp Leu Gly Arg 50 55 60 29 61 PRTArabidopsis thaliana 29 Pro Met Asp Glu Arg Glu Leu Lys Arg Glu Arg ArgLys Gln Ser Asn 1 5 10 15 Arg Glu Ser Ala Arg Arg Ser Arg Leu Arg LysGln Gln Glu Cys Glu 20 25 30 Glu Leu Ala Gln Lys Val Ser Glu Leu Thr AlaAla Asn Gly Thr Leu 35 40 45 Arg Ser Glu Leu Asp Gln Leu Lys Lys Asp CysLys Thr 50 55 60 30 61 PRT Arabidopsis thaliana 30 Pro Lys Asp Asp LysGlu Ser Lys Arg Glu Arg Arg Lys Gln Ser Asn 1 5 10 15 Arg Glu Ser AlaArg Arg Ser Arg Leu Arg Lys Gln Ala Glu Thr Glu 20 25 30 Glu Leu Ala ArgLys Val Glu Leu Leu Thr Ala Glu Asn Thr Ser Leu 35 40 45 Arg Arg Glu IleSer Arg Leu Thr Glu Ser Ser Lys Lys 50 55 60 31 61 PRT Arabidopsisthaliana 31 Pro Gln Asn Glu Arg Glu Leu Lys Arg Glu Arg Arg Lys Gln SerAsn 1 5 10 15 Arg Glu Ser Ala Arg Arg Ser Arg Leu Arg Lys Gln Ala GluThr Glu 20 25 30 Glu Leu Ala Arg Lys Val Glu Ala Leu Thr Ala Glu Asn MetAla Leu 35 40 45 Arg Ser Glu Leu Asn Gln Leu Asn Glu Lys Ser Asp Lys 5055 60 32 61 PRT Arabidopsis thaliana 32 Pro Gln Asn Glu Arg Glu Leu LysArg Glu Lys Arg Lys Gln Ser Asn 1 5 10 15 Arg Glu Ser Ala Arg Arg SerArg Leu Arg Lys Gln Ala Glu Ala Glu 20 25 30 Glu Leu Ala Ile Arg Val GlnSer Leu Thr Ala Glu Asn Met Thr Leu 35 40 45 Lys Ser Glu Ile Asn Lys LeuMet Glu Asn Ser Glu Lys 50 55 60 33 40 DNA Arabidopsis thaliana 33ggatcctgtc gtggggacac gtggcatacg aggcgaattc 40 34 40 DNA Arabidopsisthaliana 34 ggatcctgtc ggggacacgt ggcgctaacg aggcgaattc 40 35 40 DNAArabidopsis thaliana 35 ggatcctgtc gggacacgtg gcgcaacacg aggcgaattc 4036 40 DNA Arabidopsis thaliana 36 ggatcctgtc gggacacgtg gcccacccggaggcgaattc 40 37 40 DNA Arabidopsis thaliana 37 ggatcctgtc gggacacgtggcacaaatag aggcgaattc 40 38 40 DNA Arabidopsis thaliana 38 ggatcctgtcgtcaatggac acgtggctag aggcgaattc 40 39 40 DNA Arabidopsis thaliana 39ggatcctgtc gtcggacacg tggcacgaag aggcgaattc 40 40 40 DNA Arabidopsisthaliana 40 gaattcgcct cgacaggaca cgtggcacgc gacaggatcc 40 41 40 DNAArabidopsis thaliana 41 ggatcctgtc gatcaatgga cacgtggcag aggcgaattc 4042 40 DNA Arabidopsis thaliana 42 gaattcgcct cggtgacacg tggcttgaccgacaggatcc 40 43 40 DNA Arabidopsis thaliana 43 ggatcctgtc ggaagtggtgacacgtggcg aggcgaattc 40 44 40 DNA Arabidopsis thaliana 44 gaattcgcctcaagaggtga cacgtggcac gacaggatcc 40 45 40 DNA Arabidopsis thaliana 45ggatcctgtc gcgacacgtg gctgttagtg aggcgaattc 40 46 40 DNA Arabidopsisthaliana 46 gaattcgcct ctaaggaaca cgtggcccgc gacaggatcc 40 47 40 DNAArabidopsis thaliana 47 gaattcgcct ccgggcggaa cacgtggcac gacaggatcc 4048 40 DNA Arabidopsis thaliana 48 ggatcctgtc gcgtgggtac acgtggcccgaggcgaattc 40 49 40 DNA Arabidopsis thaliana 49 ggatcctgtc gcggtctttatgacacgtgg aggcgaattc 40 50 40 DNA Arabidopsis thaliana 50 gaattcgcctcggacacgtg tsgcgatccc gacaggatcc 40 51 40 DNA Arabidopsis thaliana 51gaattcgcct ctaaggcggg acacgtgtsc gacaggatcc 40 52 39 DNA Arabidopsisthaliana 52 gaattcgcct ctgacactgt cagtcccacg acaggatcc 39 53 40 DNAArabidopsis thaliana 53 gaattcgcct cggggccacg tggcttccgc gacaggatcc 4054 40 DNA Arabidopsis thaliana 54 gaattcgcct cttcgatggc cacgtggcgcgacaggatcc 40 55 40 DNA Arabidopsis thaliana 55 gaattcgcct cttaagtggccacgtggcgc gacaggatcc 40 56 40 DNA Arabidopsis thaliana 56 gaattcgcctctcacgaggc cacgtggcac gacaggatcc 40 57 40 DNA Arabidopsis thaliana 57gaattcgcct ccgtggcgcc acgtggccgc gacaggatcc 40 58 40 DNA Arabidopsisthaliana 58 gaattcgcct caatgcaccg ccacgtggcc gacaggatcc 40 59 40 DNAArabidopsis thaliana 59 gaattcgcct ccctgactgc cacgtggcac gacaggatcc 4060 40 DNA Arabidopsis thaliana 60 gaattcgcct ccaagcgttc gccacgtggcgacaggatcc 40 61 40 DNA Arabidopsis thaliana 61 gaattcgcct ctttgtccacgtggcccacc gacaggatcc 40 62 40 DNA Arabidopsis thaliana 62 gaattcgcctctagaccgtc cacgtggccc gacaggatcc 40 63 40 DNA Arabidopsis thaliana 63gaattcgcct ctaccacgtg gcacaccgtc gacaggatcc 40 64 40 DNA Arabidopsisthaliana 64 ggatcctgtc ggctaccacg tggcaagaag aggcgaattc 40 65 40 DNAArabidopsis thaliana 65 gaattcgcct cccttagcac cacgtggcac gacaggatcc 4066 40 DNA Arabidopsis thaliana 66 ggatcctgtc ggttcgatga cgtggcgaggaggcgaattc 40 67 40 DNA Arabidopsis thaliana 67 ggatcctgtc ggcttgatgacgtggccacg aggcgaattc 40 68 40 DNA Arabidopsis thaliana 68 gaattcgcctccttgatgac gtggcaccac gacaggatcc 40 69 40 DNA Arabidopsis thaliana 69ggatcctgtc gtggctgacg tggcactagg aggcgaattc 40 70 40 DNA Arabidopsisthaliana 70 ggatcctgtc ggcgcgtggt gacgtggccg aggcgaattc 40 71 40 DNAArabidopsis thaliana 71 ggattctgtc gattcggtga cgtgtcccgg aggcgaattc 4072 40 DNA Arabidopsis thaliana 72 gaattcgcct ctggctgctg acgtgtccccgacaggatcc 40 73 40 DNA Arabidopsis thaliana 73 ggatcctgtc gacgtggcaacttgaacgcg aggcgaattc 40 74 40 DNA Arabidopsis thaliana 74 gaattcgcctcgccctgaag tggacagcgc gacaggatcc 40 75 40 DNA Arabidopsis thaliana 75gaattcgcct cgccctgaag tggacagcgc gacaggatcc 40 76 40 DNA Arabidopsisthaliana 76 gaattcgcct cccgtccgcg tggcagcagc gacaggatcc 40 77 40 DNAArabidopsis thaliana 77 ggatcctgtc ggcgcgtggt gacgtggccg aggcgaattc 4078 40 DNA Arabidopsis thaliana 78 ggatcctgtc gcgtgggtac acgtggcccgaggcgaattc 40 79 40 DNA Arabidopsis thaliana 79 ggatcctgtc gcgtgccacgtgtcctgtcg aggcgaattc 40 80 60 PRT Arabidopsis thaliana 80 Leu Glu LysVal Val Glu Arg Arg Gln Lys Arg Met Ile Lys Asn Arg 1 5 10 15 Glu SerAla Ala Arg Ser Arg Ala Arg Lys Gln Ala Tyr Thr Leu Glu 20 25 30 Leu GluAla Glu Ile Glu Ser Leu Lys Leu Val Asn Gln Asp Leu Gln 35 40 45 Lys LysGln Ala Glu Ile Met Lys Thr His Asn Ser 50 55 60 81 60 PRT Arabidopsisthaliana 81 Val Glu Lys Val Val Glu Arg Arg Gln Arg Arg Met Ile Lys AsnArg 1 5 10 15 Glu Ser Ala Ala Arg Ser Arg Ala Arg Lys Gln Ala Tyr ThrVal Glu 20 25 30 Leu Glu Ala Glu Val Ala Lys Leu Lys Glu Glu Asn Asp GluLeu Gln 35 40 45 Arg Lys Gln Ala Arg Ile Met Glu Met Gln Lys Asn 50 5560 82 60 PRT Arabidopsis thaliana 82 Leu Glu Lys Val Ile Glu Arg Arg GlnLys Arg Met Ile Lys Arg Arg 1 5 10 15 Glu Ser Ala Ala Arg Ser Arg AlaArg Lys Gln Ala Tyr Thr Met Glu 20 25 30 Leu Glu Ala Glu Ile Ala Gln LeuLys Glu Leu Asn Glu Glu Leu Gln 35 40 45 Lys Lys Gln Val Glu Ile Met GluLys Gln Lys Asn 50 55 60 83 60 PRT Arabidopsis thaliana 83 Leu Glu LysVal Ile Glu Arg Arg Gln Arg Arg Met Ile Lys Asn Arg 1 5 10 15 Glu SerAla Ala Arg Ser Arg Ala Arg Lys Gln Ala Tyr Thr Leu Glu 20 25 30 Leu GluAla Glu Ile Glu Lys Leu Lys Lys Thr Asn Gln Glu Leu Gln 35 40 45 Lys LysGln Ala Glu Met Val Glu Met Gln Lys Asn 50 55 60

What is claimed is:
 1. An isolated nucleic acid molecule encoding theAbscisic acid responsive element-binding factor 2 (ABF2) having theamino acid sequence of SEQ ID NO:4.
 2. The isolated nucleic acidmolecule of claim 1, wherein the nucleic acid molecule is a messengerRNA molecule.
 3. The isolated nucleic acid molecule of claim 1, whereinthe nucleic acid molecule is a cDNA having the nucleotide sequence ofSEQ ID NO:3.